Our Science – Kashlev Website
Mikhail Kashlev, Ph.D.
My Section investigates the mechanisms regulating transcription elongation by E. coli RNA polymerase and the yeast RNA polymerase II (Pol II). We are strongly convinced that the single cell organisms (E. coli and the budding yeast) will continue to be most instrumental in understanding the basic principles of induced and spontaneous mutagenesis of DNA, which lead to tumorogenesis. Having this thought in mind, we investigate the following subjects: (1) mechanism of transcription-coupled DNA damage repair, (2) mechanisms employed by Pol II for transcription across bulky DNA lesions such as pyrimidine dimers and cyclopurines, (3) mechanisms regulating transcriptional fidelity.
We are also interested in understanding the basic mechanism of transcriptional pausing, arrest and termination using RNA polymerase from E. coli. On this avenue, we employ single molecule, pre steady-state kinetic approaches, high-resolution RNA-seq, Small Angle X-Ray Scattering (SAXS) and X-ray crystallography in addition to the methods of conventional biochemistry.
Direct assessment of transcription fidelity by high-resolution RNA sequencing (RNA-seq)
Cancerous and aging cells have long been thought to be impacted by transcription errors that cause genetic and epigenetic changes. Until now a lack of methodology for directly assessing such errors hindered evaluation of their impact to the cells. We have developed a high-resolution Illumina RNA-seq method that can assess non-coded base substitutions in mRNA at 10.000-100.000 per base frequencies in vitro and in vivo. Statistically reliable detection of changes in transcription fidelity assures that the RNA-seq can analyze transcription errors in a large number of genomic sites. A combination of the RNA-seq and biochemical analyses of the positions for the errors revealed two sequence-specific mechanisms that increase transcription fidelity by Escherichia coli RNA polymerase: (i) enhanced suppression of nucleotide misincorporation that improves selectivity for the cognate substrate, and (ii) increased backtracking of the RNA polymerase that decreases a chance of error propagation to the full-length transcript after misincorporation and provides an opportunity to proofread the error. The high-resolution RNA-seq is adoptable to a genome-wide assessment of transcription fidelity and post-transcriptional RNA editing far beyond E. coli cells.
Publications: Imashimizu et al., Nucl Acids Res (2013): doi:10.1093/nar/gkt698
Mechanisms of Transcription Through Chromatin by RNA Polymerase II and III
See cover picture in April issue of Molecular Cell
This project addresses the intriguing question of how eukaryotic RNA polymerases transcribe through chromatin. The project involves development of the minimal in vitro system for transcription through nucleosome by Pol II and Pol III from S. cerevisiae. RNAP from E. coli, which behaves very similarly to the eukaryotic enzyme in nucleosomal transcription, is used in this project as an easy-handled prototype of Pol II.
Is it related at all to cancer in humans? Yes, it is.
A central characteristic of cancer is deregulation of transcription control leading to activation of expression of growth-promoting genes, as well as silencing of genes with the tumor suppressor functions. Importantly, mutations found in tumor cells cannot alone explain the complexity of the change in pattern of gene expression. Epigenetic changes in the transcribed regions, such as DNA methylation, covalent modifications of the histones, and ATP-dependent chromatin remodeling have been recently identified as key universal components of the transformation process. Therefore, epigenetic changes are considered promising targets for new anti-cancer drugs. However, development of the novel therapies is significantly hampered by the complexity of the interplay between the different pathways of epigenetic modifications. Rapid progress in this field depends on the understanding of the molecular mechanism of action of each of the epigenetic modifications, and establishment of hierarchy of the events ultimately leading to alterations of gene expression.
To predict the effect of a certain epigenetic modification on the level of expression of a given gene, it is necessary to understand how the changes in chromatin, caused by remodeling and modification, affect transcription. Pol II transcribes templates packaged into the nucleosomes. Several lines of experimental evidence suggest that chromatin remodeling and modification occurs during promoter activation, and is associated with transcription elongation. It is not known if any chromatin remodeling and modification events are generally required for efficient elongation, and which play the gene-specific regulatory roles. To understand the molecular mechanism of transcription on chromatin templates by Pol II in vivo, we determine how the polymerase transcribes through a single nucleosome and short nucleosomal arrays in vitro.
Publications: Kireeva et al., Molecular Cell, 2002; Walter et al., JBC, 2003; Kireeva et al., Methods in Enzymology, 2003; Walter et al., Methods in Enzymology, 2003; 2004); Studitsky et al., TIBS, 2004; Kireeva et al., Molecular Cell, 2005; Galburt et al., Nature 2007
Fidelity of Transcription
Mechanism for transcription fidelity has not been identified. It's commonly accepted that transcription makes less errors than translation, but more errors than DNA replication. Although in vitro RNA polymerase makes one error per ~100000 bases, the rate of mis-transcription in vivo is unknown. Several attempts to measure the error rate in bacterial and yeast cells produced no definite results and numerous genetic searches for mutations altering RNAP fidelity failed. Our interest in this theme derives from a simple thought that mutations in the putative 'proofreading center' of Pol II may increase the level of mis-transcription above that of mis-translation, and translation of the erroneous mRNA may lead to the enzymes possessing an altered catalytic activity. In this scenario, the appearance of the error-prone DNA polymerase or DNA repair enzyme may lead to sudden burst of the mutation rate in the individual cell, causing the 'error catastrophe'. Transcriptional errors may also play roles in tumorogenesis, process of aging in humans, adaptive mutagenesis, and mutagenic evolution of retroviruses and retrotransposons. This project relies on a close cooperation with Jeff Strathern's and Ding Jin's sections of GRCBL. Our part in this collaboration involves biochemical analysis of the fidelity mechanism by Pol II.
Publications: Malagon et al., Genetics 2006; Kireeva et al., Molecular Cell 2008; Kireeva et al., METHODS 2009; Walmacq et al., J. Biol. Chem. 2009; Kireeva et al., PNAS 2009
Mechanism of Intrinsic Transcription Termination in E. coli
In the process of intrinsic transcription termination elongation complex dissociates as it passes across a DNA sequence that encodes a GC-rich hairpin in the RNA followed by a series of U residues. We investigate the mechanism of termination in E. coli in the highly purified immobilized in vitro transcription system. We address the mechanism of destabilizing action of the hairpin, the input of oligo-U sequence to the destabilization, and the integration of both signals in the termination process.
Publications: (Kashlev and Komissarova, J. Biol. Chem., 2002; Komissarova et al., Molecular Cell, 2002; Komissarova et al., Methods in Enzymology, 2003)
What Determines High Processivity of RNA Polymerase in Transcription Elongation
We address the structural basis for high processivity of transcription by studying the role of RNA and DNA in the stability of TECs. We found that formation of a stable TEC by E. coli RNAP depends on two distinct nucleic acid components: an 8-9 nt transcript-template hybrid, and a DNA duplex immediately downstream from the hybrid. For the Pol lI, only the 8-9 nt RNA:DNA hybrid is required
Publications: (Kireeva et al., JMB, 2000; Kireeva et al., JBC, 2000; Komissarova et al., Molecular Cell, 2002)
Crystallization of Elongation Complex of E. coli RNA polymerase with Nus Factors
The project aims crystallization of TEC of E. coli RNAP alone, and in combination with the transcription elongation factors NusA, NusG, NusB, and NusE. In the future, this project will include co-crystallization of the TEC with a variety of modified substrate NTPs in the active center of the enzyme to address the catalytic mechanism of RNAP.
Publications: (Komissarova et al., Methods in Enzymology, 2003)
Exploring DNA-protein and DNA-RNA Interactions in Elongation Complex of RNA Polymerase
To analyze the long-distance interactions between RNAP and DNA, which may affect the stability of TECs, we modified a specific site in the RNAP with Fe2+-BABE molecule. This agent is capable of generating hydroxyl radicals that locally break the DNA strands. This approach allows us: (1) to address the global DNA folding in TEC and its role in the complex stability and catalytic activity, and (2) to probe the DNA arrangement in the TEC interior, which is not accessible with other chemical probes. We also explore the highly sensitive method of iodine-125 radioprobing to study the geometry of nucleic acids in the TEC interior and to detect rearrangements of that geometry throughout the catalytic cycle of RNAP.
Publications: (Karamychev et al., Methods in Enzymology, 2003)
Philip J. Brooks, NIAAA-NIH, Bethesda MD
Donald Court, NCI-Frederick, MD
Xinhua Ji, NCI-Frederick, MD
Ding Jin, NCI-Frederick, MD
Shalini Oberdoerffer, NCI-Frederick, MD
Thomas Schneider, NCI-Frederick, MD
Bruce Shapiro, NCI-Frederick, MD
Jeffrey Strathern, NCI-Frederick, MD
Yun-xing Wang, NCI-Frederick, MD
Zhi-Ming (Thomas) Zheng, NCI-Frederick, MD
John Atkins, University College Cork, Cork, Ireland
Zachary Burton, Michigan State University, MI
Carlos Bustamante, UC-Berkeley, CA
Craig Cameron, PSU, University Park, PA
Benoit Coulombe, Clinical Research Institute of Montreal, Canada
Max Gottesman, Columbia University, New York, NY
Taku Oshima, Nara Institute of Science and Technology, Ikoma, Japan
Michelle Wang, Cornell University, NY
Dong Wang, UCSD, San Diego, CA
This page was last updated on 8/15/2013.